Expression of isolated DNA sequences in a plant host is dependent upon the presence of operably-linked regulatory elements that are functional within the plant host. Choice of the regulatory sequences will determine when and where within the organism the isolated DNA sequence is expressed. Where continuous expression is desired throughout the cells of a plant, a constitutive promoter is utilized. In contrast, where gene expression in response to a stimulus is desired, an inducible promoter is the regulatory element of choice. Where expression in specific tissues or organs is desired, tissue-preferred promoters and/or terminators are used. That is, these regulatory elements can drive expression preferentially in specific tissues or organs. Additional regulatory sequences upstream and/or downstream from the core sequences can be included in expression cassettes of transformation vectors to bring about varying levels of expression of isolated nucleotide sequences in a transgenic plant. See, for example, U.S. Pat. No. 5,850,018.
Plants have two basic growth modes during their life cycles: vegetative growth and reproductive growth. The above-ground vegetative growth of the plant develops from the apical meristem. This vegetative meristem gives rise to all of the leaves that are found on the plant. The plant will maintain its vegetative growth pattern until the apical meristem undergoes a change. This change alters the identity of the meristem from a vegetative to an inflorescence meristem. The inflorescence meristem produce small leaves before it next produces floral meristems. It is the floral meristem from which the flower develops.
The floral meristem undergoes a series of developmental changes that eventually give rise to the four basic structures of the flower: sepals, petals, stamens and carpels. Each of these structures is derived sequentially from a whorl that develops from the floral meristem. The first whorl develops into the sepals of the plant. The second whorl develops into petals. The third and fourth whorls define the stamen (male reproductive organs) and carpel (female reproductive organs), respectively.
From a genetic perspective, two changes that control vegetative and floral growth are programmed into the plant. The first genetic change involves the switch from the vegetative to the floral state. If this genetic change is not functioning properly, then flowering will not occur. The second genetic event follows the commitment of the plant to form flowers. The observation that the organs of the plant develop in a sequential manner suggests that a genetic mechanism exists in which a series of genes are sequentially turned on and off. This switching is necessary for each whorl to obtain its final unique identity.
A series of Arabidopsis mutants have been identified in which normal flowers are replaced with structures that resemble inflorescence meristems and the shoots that normally develop from them. One such mutant is LEAFY. This mutant does not contain any normal flowers. Instead, the early flower structures that develop appear as inflorescence shoots, whereas the later flowers partially resemble normal flowers. These later-developing flowers contain sepal and carpel-like structures; however, they lack petals and stamens. This suggests that LEAFY has two functions: committing the plant to floral meristem development, and defining petals and stamens.
Another Arabidopsis gene affecting flower intiation and development is APETALA1, also known as AP1. The AP1 gene product has been classified as a MADS protein and acts as a transcription factor. Specific motifs within MADS proteins regulate binding to promoters of other genes involved in floral development. Interactions among the MADS proteins are also possible. Conserved regions include the MADS domain, the K-box, the I-region, and the C-region. Within the general class of MADS proteins are several families. AP1 falls within the SQUA family, members of which generally are involved in both floral meristem identity and in floral organ development. This is reflected in spatial expression differences; e.g., AP1 mRNA is observed throughout the floral meristem during early flower development, but only in the outer two whorls as sepal and petal development is initiated. AP1 acts within a complex network of regulatory genes; it appears to be positively regulated by LFY, and its expression is also dependent on flowering-related genes such as FT and FD. AP1 expression is negatively regulated by PISTILLATA (PI) and an interacting protein, APETALA3 (AP3). (See, Sundström, et al., Plant Journal 46:593-600 (2006); Jang, et al., Plant Cell Physiol. 43(1)230-238 (2002); Reichmann and Meyerowitz, Biol. Chem. 378:1079-1101 (1997); Mandel, et al., Nature 360: 273-277 (1992); Coen and Meyerowitz, Nature 353:31-37 (1991)) Flowers of APETALA1 (AP1) mutants are not altered as dramatically as LEAFY mutants. These mutants express a partial inflorescence meristem phenotype where secondary floral meristems appear in the axis region of the sepal. But when the APETALA1 and LEAFY mutants are combined, the flowers appear as an inflorescence shoot. The snapdragon analog to the APETALA1 gene, SQUAMOSA, is much more severe, and the flowers appear as inflorescence shoots. APETALA1 also affects the normal development of sepals and petals.
Cloning of the Arabidopsis genes involved in the commitment to flowering and the genes controlling flower organ development has been achieved either by heterologous probing with snapdragon genes or by transposon tagging.
AP1 genes have also been identified in maize. See, for example, Münster et al., Maydica 47:287-301 (2002) and GenBank accession ZMA430695.
Maize is a monocotyledonous plant species and belongs to the grass family. It is unusual for a flowering plant as it has unisexual inflorescences The male inflorescence (tassel) develops in a terminal position, whereas the female inflorescences (ears) grow in the axil of vegetative leaves. The inflorescences, as typical for grasses, are composed of spikelets. In the case of maize each spikelet contains two florets (the grass flower) enclosed by a pair of bracts (inner and outer glume).
The grass flower is sufficiently different from a typical angiosperm flower. The lafter is composed of concentric whorls of sepals and petals enclosing whorls of stamens and pistils. The homologies of the angiosperm flower-tissues to those of the grass floret have long been debated.
According to the invention, developmentally-specific regulatory sequences are disclosed which enable the transcription of genes during the critical time of inflorescence development, preferably in early flowering tissues such as meristems, to manipulate traits such as flowering time, flower initiation, and meristem development in plants.
Isolation and characterization of promoters and terminators active in early stages of flower development can serve as regulatory elements for expression of isolated nucleotide sequences of interest in a flowering-preferred manner and are useful for manipulations targeting improved flowering traits in plants.